Uncomplicated malaria Although long-lived classical MBCs (CD19+/CD20+/CD21+/CD27+/CD10-) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical memory B cells (MBCs) (CD19+/CD20+/CD21-/CD27-/CD10-). Similar expansions of atypical MBCs have been described for a variety of chronic infectious diseases including HIV-AIDS. At present, the function of atypical MBCs in malaria is not known nor are the factors that drive their differentiation. We recently carried out studies to establish the relationship between atypical MBC and classical MBCs and provided evidence that atypical MBCs and classical MBCs have undergone the same number of cell divisions and that the antibody V gene repertoires and somatic hypermutation rates are indistinguishable suggesting a common developmental history. We showed that atypical MBCs have lost two key adaptive immune cell functions, namely the ability to signal through the BCR in response to soluble antigen and to differentiate into Ab secreting cells under a variety of conditions. To better understand the relationship between B cell subsets in malaria, we established a collaboration with Dr. Jun Zhu (NHLBI), an expert in RNAs as regulators of cellular differentiation, to carry out single cell RNA seq analysis of B cells from adults from malaria-endemic Mali. We have thus far obtained high quality RNA sequences and are in the process of analyzing these data. Our goal is to determine which RNAs are necessary for the differentiation of atypical MBCs with a view toward understanding their function. To better understand the relationship of atypical MBCs in malaria and in other chronic infectious diseases we collaborated with Dr. Susan Moir (NIAID) to compare the single cell RNA expression profile from B cells from high-viremic HIV-infected individuals. These data are being analyzed along with the RNA-seq data from malaria exposed individuals. We recently completed a study to define the factors that drive the differentiation of human tonsil B cells to acquire the phenotype of atypical MBCs in vitro. We determined that predominantly naive B cells gave rise to T-bet + aMBCs. Key factors that triggered differentiation included: the form in which antigen was presented to B cells with presentation on membranes being most effective, and the presence of gamma IFN and the TLR9 agonist CpG. Provided with these stimuli naive B cells expressed a variety of genes associated with atypical MBCs including T-bet and FcRL5 and lost their ability to be triggered through the BCR. A manuscript describing these findings is under review. Over the last year we have also carried out an in-depth analysis of the ability of atypical MBCs from Malian adults to capture, process and present antigen to helper T cells. We discovered that atypical MBCs are able to robustly capture and process antigen and remarkably the mechanisms that underlie these processes resemble those of germinal center B cells not conventional MBCs. This finding has led to the hypothesis that the function of atypical MBCs is to interact with and regulate T cell functions during malaria. We are testing this novel hypothesis using human Tfh cells and atypical MBCs in vitro. We also described the antibody V gene repertoire in response to malaria in infants and young children in Mali in collaboration with Dr. Ning Jiang (University of Texas, Austin). We used a sequencing method based on the use of molecular identifiers in combination with a clustering method that revealed more than 80% of the antibody diversity from as few as 1,000 B cells. We discovered high levels of somatic hypermutation in infants as young as three months old that gradually increased with age and stabilized in toddlers. Our results highlighted the vast potential antibody repertoire diversification in infants that had not been previously recognized and could have a profound impact on vaccination strategies in children. We also collaborated on malaria projects with NIAID investigators to determine the role of NK cells in inhibiting Pf growth in infected red blood cells by ADCC and to evaluate the efficacy of a malaria vaccine in Aotus monkeys. Cerebral malaria In recent years we have used a mouse model of cerebral malaria (CM), termed experimental CM (ECM) to identify potential adjunctive therapies that could be used in African children. ECM exhibits many of the same features in CM including parasite infected red blood cells in the brain vasculature, brain swelling, hemorrhaging and breakdown of the blood brain barrier. In addition accumulation of CD8+ T cells in the brain has been shown to play a critical role in ECM disease. However, at present a role for CD8+ T cells in CM has not been rigorously investigated. We have tested a number of inhibitors of T cell metabolism as adjunctive therapy for CM in collaboration with Dr. Jonathan Powell (Johns Hopkins University). Remarkably we found that a glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), rescued mice from ECM, when administered late in the infection when mice already showed neurological signs of the disease. At the time of treatment mice were suffering blood-brain barrier dysfunction, brain swelling and hemorrhaging accompanied by accumulation of parasite-specific CD8+ effector T cells and infected red blood cells in the brain and perturbation of brain metabolism. Remarkably, DON-treatment restored blood-brain barrier integrity, reduced brain swelling, decreased the function of activated effector CD8+ T cells in the brain and returned brain metabolites to uninfected levels. Our goal over the last year was to better understand the similarities and differences in the pathology associated with CM in children in Malawi and ECM in mice to allow us to better evaluate DON as an adjunctive therapy in CM. To this end we established a collaboration with Dr. Terrie Taylor (Michigan State University), an expert in CM in children who heads a pediatric clinic in Malawi that treats children with CM. We described the immune infiltrates into the brains of children who died of CM using multiplex immunohistochemistry analysis of brain sections. We have now established for the first time that CD8+ T cells sequester along the abluminal face of blood vessels in the brains of children with CM. This finding opens up a new world of possible adjunctive therapies and validates the use of DON as CM therapy. We have also established a collaboration with Dr. Dima Hammoud (NIAID), an expert in the application of MRI and PET imaging to the study of brain infections to image brains of mice with CM. We used longitudinal MR imaging to visualize brain pathology in ECM and the impact of DON on disease progression in mice. For the first time, we demonstrated in vivo the reversal of disease markers in symptomatic infected mice following DON treatment, including resolution of edema and blood brain barrier disruption, findings usually associated with fatal outcome in children and adults with CM. Our results support the premise that DON is a potential adjunctive treatment that could rescue children and adults from fatal CM.